Porphyron based neuton capture agents for cancer therapy

ABSTRACT

The invention describes the synthesis of a panel of novel carbon-carbon linked carboranyl-containng 5,10,15,20-tetraphenylporphyrins bearing 25-44% boron by weight. Preliminary in vitro evaluation of several of these compounds, using both rat and human brain tumor cell lines, shows that these compounds display very low cytotoxicities, are capable of delivering substantial amounts of boron intracellularly, and are retained for long periods of time.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with Government support under Grant No.98ER62633, awarded by the Department of Energy. The Government hascertain rights to this invention.

FIELD OF THE INVENTION

[0002] The field of the invention is cancer therapy.

BACKGROUND OF THE INVENTION

[0003] Boron neutron capture therapy (BNCT) is a bimodal modality forcancer treatment consisting on the irradiation of ¹⁰B-rich tumors withlow-energy (thermal) neutrons, with subsequent production of high linearenergy transfer particles, ⁴He²⁺ (alpha-particle) and ⁷Li³⁺, which causesevere damage to tumor cells through ionization processes; see Barth,Soloway, Fairchild et al., infra; Barth, Soloway, Goodman et al., infra.Because the cytotoxic ions produced in the nuclear reaction have alimited distance of travel in tissue (approximately one cell diameter,5-9 μm), the success of this modality for cancer therapy depends on theselective uptake of boron in the tumor cells; see Hawthorne, (1998),infra; Hawthorne, (1993). infra. Malignant brain tumors are responsiblefor more than 10,000 deaths per year in the United States, and BNCT isspecially attractive as the treatment because it potentially targets anddestroys malignant cells in the presence of normal cells, thus,preventing undesirable side effects common in other types of treatments.In addition, BNCT has advantages over photoradiation therapy in thatneutron beams can penetrate upwards of ten times deeper, to reachdeep-seated tumor sites (6-7 cm).

[0004] In recent years, several research groups have developed a varietyof new ¹⁰B carriers with improved tumor selectivity over the two boronneutron capture agents currently undergoing clinical trials in the U.S.,Europe, and Japan, for the treatment of patients with glioblastomas andmelanomas disodium mercapto-closo-dodecaborate (BSH) andL-4-dihydroxy-borylphenylalanine (BPA). See Kageji, et al., infra.Pignol, et al., infra; Elowitz, et al., infra. To date, of all the newboron-delivery agents, porphyrins are particularly promisingtumor-selective compounds because of their natural tendency toaccumulate in neoplastic tissue; see Bonnett, infra. This property ofporphyrins provides the basis for their use in another therapeuticmethod, the photodynamic therapy (PDT) of tumors; see Schnitmaker, etal., infra; Dougherty, et al., infra. PDT relies on the selective uptakeof a photosensitizer in tumor tissues, followed by generation of singletoxygen and other cytotoxic species upon irradiation with red light.Photofrin®, a porphyrin derivative and only FDA-approved PDT drug hasbeen used to treat thousands of patients in Canada, Europe, Japan andU.S. with early and advanced stage cancer of the lung, digestive tract,and genitourinary tract. In addition to necrosis as the result ofoxidative damage, it has been recently shown that some porphyrins alsoinduce apoptosis (programmed cell death), either upon irradiation withlight (particularly at low light doses), or by accumulation of high druglevels in tissues, in the dark.; see Luo, Chang, et al., infra; Luo, Ke,et al., infra. The ability of porphyrin-PDT to induce apoptosis may alsobe an important element for the success of both PDT and BNCT modalitiesfor cancer treatment. Photofrin® presents the disadvantages of being acomplex mixture of compounds of variable composition. Thus, activeresearch in the area of development of new and highly efficient PDTphotosensitizers is underway. Also, boron-containing porphyrinderivatives that selectively localize in tumor cells have potential PDTapplications; see Hill, Kahl, Stylli et al., infra.

[0005] Porphyrins and their diamagnetic metal complexes are highlyfluorescent. This provides a means for detection of tumor cells and forinvestigation of the ¹⁰B localization in tumors and surrounding tissues;see Mang et al., infra.

[0006] Intracellular boron distribution and quantification playimportant roles in determining the dose and length of a neutronradiation period, as well as the success of BNCT treatment; see Nigg etal., infra. The intracellular localization of porphyrins is highlydependent upon their physicochemical properties, including structuralfeatures such as nature of peripheral side chains, hydrophobicity,charge, molecular weight, and amphiphilic character; see Woodburn,Vardaxis, et al., infra. Certain porphyrins have been reported to havethe ability to target the nuclei of tumor cells and causing DNA damagethrough intercalation, or binding to DNA; see Munson and Fiel, infra;Schneider and Wang, infra; Ding et al., (1991), infra; Sari et al.,infra; Penning et al., infra. Therefore, the newly developed BNCT agentswill display an appropriate balance between hydrophobicity andhydrophilicity. This property will give adequate solubility in aqueousor biological media, and enhanced interaction with cell membranes.

[0007] Although BSH and BPA have been shown to be safe and efficaciousin animal models, BSH is reported to be sensitive to air-oxidation(Tolpin et al., infra) and both BSH and BPA have only moderateselectivity for tumor cells and low retention times in tissues (Capalaet al., infra). The ultimate success of BNCT is dependent upon whetheradequate concentrations of boron-containing capture agents andlow-energy neutrons can be selectively and effectively delivered totumor cells. Since selective production of epithermal neutrons with highbeam quality has been achieved by modern nuclear reactors (such as theone available at the McClellan Nuclear Radiation Center in Sacramento),the main unsolved problem in BNCT centers on the development of neweffective ¹⁰B carriers, capable of selectively delivering substantialconcentrations of ¹⁰B atoms to tumors. In the last ten years, severalboron-containing porphyrin derivatives have been reported forapplication in BNCT and their in vitro and in vivo properties evaluated;see Hill, Kahl, Kaye et al., infra; Woodbum, Phadke et al., infra;Ceberg et al., infra; Ozawa et al., infra; Miura, Micca, et al., infra;Matsumura et al., infra. These studies reveal that boronated porphyrinsaccumulate within cells of glioma models to a much greater extent, andare retained for longer periods of time, than do BSH and BPA. Inaddition, along with low toxicities and favorable intracellularbiodistributions, significantly higher tumor porphyrin ratios relativeto blood and normal tissue are found for some of these boronatedporphyrins. To date, amongst the porphyrin-based BNCT drugs reported, invivo investigations with BOPP (a protoporphyrin-IX derivative) andNiTCP-H (a meso-tetraphenylporphyrin derivative) are the most promising;see Kahl and Koo, infra; Miura, Gabel et al., infra. BOPP contains fourcarboranyl residues linked by ester bonds to the porphyrin macrocycle.In vivo cleavage of such linkages has been accounted for the sometimesobserved low retention times of this drug in tumor cells. The highlylipophilic NiTCP-H contains four ether-linked carboranyl moieties, andrequires the use of solubilizing agents such as Cremophor EL (apolyethoxylated castor oil) and propylene glycol as delivery vehicles;secondary effects of these solubilizing agents are not yet wellunderstood; see Woodcock et al., infra; Badary et al., infra. Otherboron-containing porphyrins are reported in the literature, but theycontain a lower percentage (5-15%) of boron by weight.

SUMMARY OF THE INVENTION

[0008] The present invention describes novel benzaldehyde intermediatesfor use the synthesis of novel carboranyl-containing5,10,15,20-tetraphenylporphyrins. The new compounds featurecarbon-carbon linkages between the carboranyl groups and the phenylgroups of the mesophenylporphyrin ring; and up to about 25-44% boron byweight. The present invention also includes methods and compositions forusing the carboranyl-containing porphyrins in cancer treatmentsinvolving boron neutron capture therapy or photodynamic therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 describes the synthesis of compounds 1-6;

[0010]FIG. 2 describes the synthesis of compounds 7-10;

[0011]FIG. 3 describes the synthesis of compounds 11-16;

[0012]FIG. 4 describes the synthesis of compounds 17-20;

[0013]FIG. 5 describes the synthesis of compounds 21-24;

[0014]FIG. 6 describes the synthesis of compounds 25-29;

[0015]FIG. 7 describes the compounds that are synthesized using asimilar methodology; and

[0016]FIG. 8 describes additional compounds that can be synthesizedusing similar methodology.

DETAILED DESCRIPTION

[0017] The present invention is directed to the synthesis and use ofporphyrin compounds, which contain carboranyl groups attached to theporphyrin compound by a carbon-carbon linkage. The porphyrin compoundgenerally corresponds to general formula I:

[0018] where M is 2H or a divalent metal ion; R1 and R2 are eachindependently hydrogen, alkyl or hydoxyalkyl; and R3 through R6 arehydrogen or a substituted phenyl group.

[0019] The substituted phenyl group corresponds to general formula II:

[0020] where R7 through R11 are hydrogen, a hydrophilic group or acarboranyl group. The carboranyl group is attached to the phenyl groupby a carbon-carbon linkage. Typically, either one or two of R7 throughR11 are hydrophilic or carboranyl groups. Examples of hydrophilic groupsinclude hydroxyl, NMe₃ ⁺, PMePh₂ ⁺, PO(OH)₂, SO₃H, COOH or NH₂.

[0021] At least one of R3 through R6 is a phenyl group of generalformula II having at least one carboranyl group attached by acarbon-carbon linkage. More preferably, two of R3 through R6 are phenylgroups of general formula II, each having at least one carboranyl group.Most preferably, R3 through R6 are all phenyl groups of general formulaII, each having at least one carboranyl group.

[0022] In a preferred embodiment of the porphyrin compound thecarboranyl group is 1-methyl-o-carboranyl or o-carboranyl. In anotherpreferred embodiment the compound is at least about 15% boron by weight;most preferably about 25% to about 44% boron by weight.

[0023] The present invention also includes a method of making theforegoing carboranyl-containing porphyrin compounds.

[0024] The first step of the method is providing a reaction mixture,which includes (1) a pyrrole or a dipyrrole, and (2) a benzaldehyde,both dissolved in a suitable solvent. The pyrrole or dipyrrole can beunsubstituted or substituted with alkyl groups; and the benzaldehyde isof general formula III

[0025] where R7 through R11 are each hydrogen, a hydrophilic group or acarboranyl group attached to the benzaldehyde by a carbon-carbonlinkage. Hydrophilic groups can include hydroxyl, NMe₃ ⁺, PMePh₂ ⁺,PO(OH)₂, P(CH₂OH)₂, SO₃ ⁻, COOH, CO₂ ⁻ or NH₂.

[0026] The next step of the method is to subject the reaction mixture toan acidic pH until the benzaldehyde and the pyrrole, or the benzaldehydeand the dipyrrole, are converted to a porphyrin compound. In onepreferred version of the method, the reaction mixture is subjected to anacidic pH by the addition of TFA. Preferred versions of the method alsoinclude oxidation of the reaction mixture with tetrachloroquinone ordichlorodicyanobenzoquinone.

[0027] Optionally, the method can include complexing the porphyrincompound with a divalent metal ion, e.g. by treating the free base ofthe porphyrin compound with zinc choride to form a Zn(II) complex.Moreover, the solubility of the porphyrin compound can be improved byforming a salt of the porphyrin compound.

[0028] The present invention also includes an intermediate for use inthe synthesis of porphyrin compounds, namely the aforementionedbenzaldehyde of general formula III

[0029] where R7 through R11 are each hydrogen, a hydrophilic group or acarboranyl group attached to the benzaldehyde by a carbon-carbonlinkage; and either one or two of R7 through R11 are carboranyl groups.

[0030] We anticipate that the carboranyl-containing porphyrins of thepresent invention will be of use in cancer treatments involving boronneutron capture therapy (BNCT). Accordingly, the present inventionincludes a method of delivering an effective amount of a neutron captureagent to tumor cells, which involves contacting the tumor cells with acarboranyl-containing porphyrin compound of the present invention. Invivo, the method includes administering the carboranyl-containingporphyrin compound to a patient, which is then selectively taken up bythe tumor cells. To be effective in BNCT, the amount of porphyrincompound taken up by the tumor cells is an amount sufficient forcytotoxicity when the tumor cells are irradiated by thermal neutrons.Preferably, the tumor cells are brain tumor or melanoma cells

[0031] Our preliminary results, described in greater detail in theExamples below, show that the carboranyl-containing porphyrins of thepresent invention will also be of use in cancer treatments involvingphotodynamic therapy (PDT). Thus, the present invention includes amethod of delivering an effective amount of a photosensitizing agent totumor cells, similar to the method for BNCT. In the PDT method, aneffective amount of the porphyrin compound is an amount sufficient forcytotoxicity when the tumor cells are irradiated by red light.

[0032] The present invention also includes compositions for use in boronneutron capture therapy comprising an effective amount of acarboranyl-containing porphyrin compound of the present invention, and apharmaceutically acceptable carrier or excipient. An effective amountfor BNCT is an amount sufficient for selective uptake, retention anddamage to tumor cells when the tumor cells are irradiated by thermalneutrons.

[0033] Similarly, the present invention includes compositions for use inphotodynamic therapy comprising the carboranyl-containing porphyrincompounds of the present invention and a pharmaceutically acceptablecarrier or excipient. The effective amount of the porphyrin compound forPDT is an amount sufficient for selective uptake, retention andcytotoxicity to tumor cells when the tumor cells are irradiated by redlight.

[0034]FIGS. 1 through 6 describe, in very few steps, the total synthesisof carboranylated 5,10,15,20-tetraphenylporphyrins. Thesemeso-phenylporphyrin compounds contain carbon-carbon linkages betweenthe carboranyl groups and the meso-phenylporphyrin ring for increasedchemical in vitro and in vivo stability over existing drugs. Inaddition, the high solubility of these new drugs in aqueous solutionallows for their easy administration into the blood stream (via aconcentrated saline solution of the drug), and avoids the use of aco-solvent. In vitro and in vivo biological activities of the new drugsshow that these new compounds are very promising drugs for both the BNCTand the PDT modalities for cancer treatment.

[0035]FIG. 7 describes the compounds that can be synthesized using thesame synthetic methodology as for compound 26 (FIG. 6). Theseunsymmetrical compounds contain carbon-carbon linked hydrophobico-carboranyl cages and hydrophilic groups (OH) to warrant solubility inaqueous solution. FIG. 8 describes additional compounds that can besynthesized using the carborane building blocks described in FIG. 5.These compounds will be analogues of compound 24, and will bear otherhydrophilic groups (neutral, positive and negative, e.g. —NMe₃ ⁺,—PMePh₂ ⁺, —P(CH₂OH)₂, SO₃ ⁻, CO₂ ⁻). In summary, all drugs in thepresent invention contain carbon-carbon linkages between the porphyrinring and the carboranyl groups, and amphiphilic properties for bothadequate solubility into the blood stream and interaction with cellmembranes.

EXAMPLES

[0036] Synthesis of Compound 1:

[0037] [4-(1-methyl-o-carboranyl)methyl]bromobenzene (1): A two-neckedround bottom flask containing 1-methyl-o-carborane (5.00 g, 31.65 mmol)in dry DME (150 mL) was cooled down to 0° C. under Argon. n-BuLi (20.0mL, 1.6 M in hexane) was added dropwise and the resulting mixture wasstirred at 0° C. for 30 minutes. A solution of4-(bromomethyl)bromobenzene (7.91 g, 31.65 mmol) in dry DME (15 mL) wasadded dropwise. After stirring at 0° C. for 10 minutes, the finalreaction mixture was warmed to room temperature and subsequentlyrefluxed for 12 hours under Argon. The solvent was then removed undervacuum and the crude solid obtained was purified by recrystallizationfrom dichloromethane/methanol to give the title compound (7.80 g, 75.4%yield) as white crystals. MS (EI) m/e 327.1 (M⁺); ¹H-NMR (CDCl₃) δ ppm:1.3-3.0 (br, 10H, BH), 2.15 (s, 3H, CH₃), 3.41 (s, 2H, CH₂), 7.06 (d,2H, ArH, J=8.1 Hz), 7.48 (d, 2H, ArH, J=8.1 Hz).

[0038] Synthesis of Compound 2:

[0039] [4-(1-methyl-o-carboranyl)methyl]benzaldehyde (2): A solution ofcompound 1 (4.00 g, 12.23 mmol) in THF (150 mL) under Argon was cooledto −78° C. (acetone/dry ice bath). n-BuLi (7.6 mL, 1.6 M in hexane) wasadded dropwise while maintaining the temperature at −78° C. The reactionmixture was stirred for 30 minutes at −7820 C. before dry DMF (5.0 mL,64.6 mmol) was slowly added. The final mixture was stirred at −78° C.for 15 minutes and then warmed up slowly to room temperature. A 2N HClsolution (150 mL) was added end the reaction mixture stirred for 2 h atroom temperature. The solution was then reduced to a volume of 200 mLand extracted with dichloromethane (4×50 mL). The organic extracts werewashed once with an aqueous saturated solution of NaHCO₃, once withwater and dried over anhydrous Na₂SO₄. After removal of the solventunder vacuum, the oily residue was purified by column chromatography onsilica gel (dichloromethane/petrbleum ether 1:1), yielding the titlecompound (2.1 g, 62% yield) as a white solid. MS (EI) m/e 276.2 (M⁺);¹H-NMR (CDCl₃) δ ppm: 1.5-3.0 (br, 10H, BH), 2.19 (s, 3H, CH₃), 3.54 (s,2H, CH₂), 7.38 (d, 2H, ArH, J=8.0 Hz), 7.89 (d, 2H, ArH, J=8.0 Hz),10.04 (s, 1H, CHO).

[0040] Synthesis of Compound 3:

[0041] meso-tetra[4(1-methyl-o-carboranyl)methylphenyl]porphyrin (3): Asolution of aldehyde 2 (1.16 g, 4.19 mmol) and freshly distilled pyrrole(0.30 mL, 4.32 mmol) in dry dichloromethane (420 mL) was purged withArgon for 15 minutes. TFA (0.25 mL, 3.15 mmol) was added to the solutionand the final mixture was stirred at room temperature under Argon for 20hours (complete disappearance of starting compound 2 by TLC). Afteraddition of p-chloranil (0.788 g, 3.14 mmol) the final reaction mixturewas stirred at room temperature for 2 hours. The solution wasconcentrated under vacuum to 200 mL, then washed once with an aqueoussaturated solution of NaHCO₃, and once with water before being driedover anhydrous Na₂SO₄. The residue obtained after removal of the solventunder vacuum was purified by column chromatography(dichloromethane/petroleum ether 1:1) and the fastest running porphyrinfraction was collected and recrystallized from dichloromethane/methanol,yielding 0.289 g (21% yield) of the title compound as purple crystals,m.p.>300° C.; MS (MALDI) m/e 1296.0 (M⁺); ¹H-NMR (CDCl₃) δ ppm: −2.80(br, 2H, NH), 1.6-3.1 (br, 40H, BH), 2.34 (s, 12H, CH₃), 3.81 (s, 8H,CH₂), 7.59 (d, 8H, ArH, J=8.0 Hz), 8.20 (d, 8H, ArH, J=8.0 Hz), 8.85 (s,8H, β-H). UV-Vis (CHCl₃) λ_(max): 418 nm (ε 467,700), 514 (16,867), 550(8,132), 590 (5,470), 646 (4,028).

[0042] Synthesis of Compound 4:

[0043] meso-tetra[4-(1-methyl-nido-carboranyl)methylphenyl]porphyrintetrapotassium salt (4): Porphyrin 3 (0.050 g, 0.0386 mmol) wasdissolved in a 3:1 mixture of pyridine and piperidine (4.0 mL), andstirred at room temperature in the dark for 36 hours, under Argon. Thesolvent was completely removed under vacuum, the residue re-dissolved ina 60% acetone aqueous solution and passed slowly through a Dowex50W2-100 resin in the potassium form. The porphyrin fraction wascollected, dried under vacuum, re-dissolved in a 30% acetone aqueoussolution and again passed through the ion-exchange resin. After removalof the solvent under vacuum, the tetraanionic porphyrin wasrecrystallized from methanol/diethyl ether, yielding 0.051 g (94% yield)of the title compound, m.p.>300° C. ¹H-NMR (CD₃COCD₃) δ ppm: −2.70 (s,2H, NH), −2.45 to −1.90 (br, 4H, BH), 0.9-2.4 (br, 32H, BH), 1.59 (s,12H, CH₃) 3.50 (s, 8H, CH₂), 7.81 (d, 8H, ArH, J=8.0 Hz), 8.08 (d, 8H,ArH, J=8.0 Hz), 8.90 (s, 8, β-H). UV-Vis (acetone) λ_(max): 420 nm (ε349,700), 516 (13,595), 554 (12,410), 594 (4,130), 650 (5,990).

[0044] Synthesis of Compound 5:

[0045] Zn(II)-meso-tetra[4-(1-methyl-o-carborane)methylphenyl]porphyrin(5): To a solution of porphyrin 3 (0.150 g, 0.110 mmol) indichloromethane (150 mL), THF (10 mL), and pyridine (0.5 mL) was addedZnCl₂.2H₂O (0.075 mg, 0.435 mmol), and the final mixture was stirred atroom temperature under Argon overnight. The mixture was then washed oncewith water, dried over anhydrous Na₂SO₄, and the solvent evaporatedunder vacuum. The residue was purified by column chromatography(dichloromethanelpetroleum ether 1:1.5), the pink color fractioncollected and recrystallized from dichloromethane/methanol, to give0.135 g (92% yield) of the title compound as purple crystals, m.p.>300°C.; MS m/e 1358.6; ¹H-NMR (CDCl₃) δ ppm: 1.6-3.0 (br, 40H, BH), 2.33 (s,12H, CH₃), 3.80 (s, 8H, CH₂), 7.57 (br s, 8H, ArH), 8.19 (br s, 8H,ArH), 8.95 (br s, 8H, β-H). UV-Vis (CHCl₃) λ_(max): 424 nm (ε 577,000),554 (20,102), 596 (6,380).

[0046] Synthesis of Compound 6:

[0047]Zn(II)-meso-tetra[4-(1-methyl-nido-carboranyl)methylphenyl]porphyrintetrapotassium salt (6): The Zn(II) complex 5 (0.075 g, 0.055 mmol) wasdissolved in a 3:1 mixture of pyridine and piperidine (4.0 mL), andstirred at room temperature in the dark for 36 hours, under Argon. Thesolvent was completely removed under vacuum, the residue re-dissolved ina 60% acetone aqueous solution and passed slowly through a Dowex50W2-100 resin in the potassium form. The porphyrin fraction wascollected, dried under vacuum, re-dissolved in a 30% acetone aqueoussolution and again passed through the ion-exchange resin. After removalof the solvent under vacuum, the tetra-anionic porphyrin wasrecrystallized from methanovdiethyl ether, yielding 0.078 g (96.x %yield) of the title compound, m.p.>300° C. ¹H-NMR (CD₃COCD₃) δ ppm:−2.48 to −1.95 (br, 4H, BH), 0.9-2.4 (br, 32H, BH), 1.59 (s, 12H, CH₃),3.50 (s, 8H, CH₂), 7.77 (d, 8H, ArH, J=8.0 Hz), 8.11 (d, 8H, ArH, J=8.0Hz), 8.92 (s, 8H, β-H). UV-Vis (acetone) λ_(max): 422 nm (ε 479,000),554 (13,870), 596 (6,595).

[0048] Synthesis of Compound 7:

[0049] [3-(1-methyl-o-carboranyl)methyl]bromobenzene (7): A two-neckedround bottom flask containing 1-methyl-o-carborane (3.00 g, 18.99 mmol)in dry THF (150 mL) was cooled down to 0° C. under Argon. n-BuLi (12.0mL, 1.6 M in hexane) was added dropwise and the resulting mixture wasstirred at 0° C. and then cooled down to −10° C. A solution of anhydrousLiI (0.350 g, 2.61 mmol) in THF (2.5 mL) was added, followed by asolution of 3-(bromomethyl)bromobenzene (5.00 g, 20.00 mmol) in THF (10mL). After stirring at −10° C. for 15 minutes, the final reactionmixture was warmed to room temperature and stirred for 12 hours underArgon. The reaction mixture was then washed with water (2×25 mL),extracted with diethyl ether (3×25 mL) and dried over Na₂SO₄. Thesolvent was then removed under vacuum and the crude solid obtained waspurified by column chromatography (silica gel, dichloromethane/petroleumether 1:9) to give the title compound (4.25 g, 65.0% yield). MS (EI) m/e327.1 (M⁺); ¹H-NMR (CDCl₃) δ ppm: 1.3-3.1 (br, 10H, BH), 2.16 (s, 3H,CH₃), 3.42 (s, 2H, CH₂), 7.13 (d, 1H, ArH, J=7.8 Hz), 7.23 (t, 1H, ArH,J=7.8 Hz), 7.33 (s, 1H, ArH), 7.47 (d, 1H, ArH, J=7.8 Hz).

[0050] Synthesis of Compound 8:

[0051] [3-(1-methyl-o-carboranyl)methyl]benzaldehyde (8): A solution ofcompound 7 (1.00 g, 3.06 mmol) in THF (25 mL) under Argon was cooled to−78° C. (acetone/dry ice bath). n-BuLi (2.0 mL, 1.6 M in hexane) wasadded dropwise while maintaining the temperature at −78° C. The reactionmixture was stirred for 30 minutes at −78° C. before dry DMF (1.0 mL,17.5 mmol) was slowly added. The final mixture was stirred at −78° C.for 15 minutes and then warmed up slowly to room temperature. A 2N HClsolution (25 mL) was added and the reaction mixture stirred for 2 h atroom temperature. The solution was then reduced to a volume of 200 mLand extracted with dichloromethane (4×50 mL). The organic extracts werewashed once with an aqueous saturated solution of NaHCO₃, once withwater and dried over anhydrous Na₂SO₄. After removal of the solventunder vacuum, the oily residue was purified by column chromatography onsilica gel (dichloromethane/petroleum ether 1:1), yielding the titlecompound (0.668 g, 79.1% yield) as a white solid. MS (EI) m/e 276.2(M⁺); ¹H-NMR (CDCl₃) δ ppm: 1.4-3.1 (br, 10H, BH), 2.19 (s, 3H, CH₃),3.55 (s, 2H, CH₂), 7.48 (d, 1H, ArH, J=7.8 Hz), 7.56 (t, 1H, ArH, J=7.8Hz), 7.70 (s, 1H, ArH), 7.85 (d, 1H, ArH, J=7.8 Hz), 10.04 (s, 1H, CHO).

[0052] Synthesis of Compound 9:

[0053] meso-tetra[3-(1-methyl-o-carboranyl)methylphenylporphyrin (9): Asolution of aldehyde 8 (0.660 g, 2.39 mmol) and freshly distilledpyrrole (0.18 mL, 2.59 mmol) in dry dichloromethane (240 mL) was purgedwith Argon for 45 minutes. TFA (0.15 mL, 1.89 mmol) was added to thesolution and the final mixture was stirred at room temperature underArgon for 18 hours. After addition of p-chloranil (0.440 g, 1.77 mmol)the final reaction mixture was stirred at room temperature for 3 hours.The organic solution was washed once with an aqueous saturated solutionof NaHCO₃, and once with water before being dried over anhydrous Na₂SO₄.The residue obtained after removal of the solvent under vacuum waspurified by column chromatography (dichloromethane/petroleum ether 1:1)and the porphyrin fraction was collected and recrystallized fromdichloromethane/methanol, yielding 0.252 g (33% yield) of the titlecompound as purple crystals, m.p.>300° C.; MS (MALDI) m/e 1296.0 (M⁺);¹H-NMR (CDCl₃) δ ppm: −2.84 (br, 2H, NH), 1.5-3.0 (br, 40H, BH), 2.20(s, 12H, CH₃), 3.74 (s, 8H, CH₂), 7.62 (d, 4H, ArH), 7.74 (d, 4H, ArH),8.05 (d, 4H, ArH), 8.18 (d, 4H, ArH), 8.84 (s, 8H, β-H). UV-Vis (CHCl₃)λ_(max): 419 nm, 516, 548, 590, 646.

[0054] Synthesis of Compound 10:

[0055] meso-tetra[3-(1-methyl-nido-carboranyl)methylphenyl]porphyrintetrapotassium salt (10): Porphyrin 9 (0.049 g, 0.0378 mmol) wasdissolved in a 3:1 mixture of pyridine and piperidine (4.0 mL), andstirred at room temperature in the dark for 36 hours, under Argon. Thesolvent was completely removed under vacuum, the residue re-dissolved ina 60% acetone aqueous solution and passed slowly through a Dowex50W2-100 resin in the potassium form. The porphyrin fraction wascollected, dried under vacuum, re-dissolved in a 30% acetone aqueoussolution and again passed through the ion-exchange resin. After removalof the solvent under vacuum, the tetraanionic porphyrin wasrecrystallized from methanol/diethyl ether, yielding 0.050 g (94% yield)of the title compound, m.p.>300° C. UV-Vis (acetone) λ_(max): 431 nm,511, 546, 590, 647.

[0056] Synthesis of Compound 11:

[0057] 4-Ethynylbenzaldehyde (11): To a solution of 4-bromobenzaldehyde(10.00 g, 54.08 mmol) and triphenylphosphine (0.500 g, 1.91 mmol) inanhydrous triethylamine (80 mL) under Angon, were addedethynyltrimethylsilane (6.00 g, 61.09 mmol) and palladium (II) acetate(0.100 g, 0.445 mmol). The final mixture was heated to reflux for 2hours, and then it was cooled down to room temperature and filtrated.The filtrate was concentrated under vacuum to a thick oil, which waspurified by column chromatography (dichloromethane/petroleum ether 1:4)and recrystallized from cold cyclohexane to give 10.5 g (96.1% yield) of4-(trimethylsilylethynyl)benzaldehyde; MS m/e 187.2 (M⁺); ¹H-NMR (CDCl₃)δ ppm: 0.27 (s, 9H, SiMe₃), 7.60 (d, 2H, ArH, J=8.1 Hz), 7.82 (d, 2H,ArH, J=8.1 Hz), 10.00 (s, 1H, CHO)]. This compound (8.00 g, 39.59 mmol)was treated with K₂CO₃ (0.500 g) in methanol (50 mL) at 25° C. for 2hours, under Argon. The solvent was removed under vacuum and the residuedissolved in dichloromethane (100 mL). This solution was washed oncewith an aqueous saturated solution of NaHCO₃ and once with water, beforebeing dried over anhydrous Na₂SO₄ and the solvent evaporated undervacuum. The yellow residue was purified by column chromatography usingdichloromethane/petroleum ether 1:4 for elution and recrystallizationfrom cold cylohexane to give 4.40 g (85.5% yied) of the title compound;MS (EI) m/e 130.0; (M⁺). ¹H-NMR (CDCl₃) δ ppm: 3.30 (s, 1H, CH), 7.64(d, 2H, ArH, J=8.1 Hz), 7.84 (d, 2H, ArH, J=8.1 Hz), 10.02 (s, 1H, CHO).

[0058] Synthesis of Compound 12:

[0059] 4-(o-carboranyl)benzaldehyde (12): BF₃.OEt₂ (0.654 g, 4.62 mmol)was added at 0° C. and under Argon, to a solution of4-ethynylbenzaldehyde (11) (6.00 g, 46.15 mmol) and 1,2-ethanedithiol(5.00 g, 53.09 mmol). This mixture was stirred at room temperature underArgon for 15 minutes. The reaction mixture was then washed once with a10% aqueous NaOH solution, and once with an aqueous saturated solutionof NaCl, before being dried over anhydrous Na₂SO₄ and the solventevaporated under vacuum. Purification of the resulting residue by columnchromatography (dichloromethane/petroleum ether 1:4) gavep-ethynylbenzyl(1,3-dithiane) (7.5 g, 79% yield) as a yellow solid [MS(EI) m/e 206.0 (M⁺); ¹H-NMR (CDCl₃) □ ppm: 3.07 (s, 1H, CH), 3.38 and3.51 (m, 2H each, CH₂CH₂), 5.61 (s, 1H, SCH), 7.42 (d, 2H, ArH, J=8.1Hz), 7.47 (d, 2H, ArH, J=8.1 Hz)]. Decaborane (3.00 g, 24.59 mmol),ethyl sulfide (5.00 g, 55.44 mmol) and dry toluene (50 mL) were combinedin a schlenk tube equipped with a stir bar. This solution was heated at40° C. for 3 hours and at 60° C. for 2 hours, and then allowed to cooldown to room temperature. To this mixture was added a solution ofp-ethynylbenzyl(1,3-dithiane) (5.00 g, 24.26 mmol) in dry toluene (10mL), and the final reaction mixture was slowly warmed up to 80° C. andstirred at this temperature for 3 days. After cooling to roomtemperature, the mixture was concentrated under vacuum and the resultingoil was dissolved in methanol (250 mL) and heated to reflux untilliberation of hydrogen ceased (approximately 60 minutes). At roomtemperature a 50% aqueous HCl solution (2 to 3 mL) was cautiously addedand the mixture was again heated to reflux until the hydrogen evolutionwas complete (approximately 30 minutes). After cooling down to roomtemperature, the reaction mixture was diluted with ethanol and excessethylsulfide was removed by ethanol-ethylsulfide co-distillation. Theremain residue was concentrated under vacuum. To a solution of theresulting residue in benzene (100 mL) at 50° C., was added 100 mL of acold 10% aqueous NaOH solution, and the final mixture stirred vigorouslyfor 15 minutes. The organic layer was separated, washed with water (3×25mL) and dried over anhydrous Na₂SO₄. After evaporation of the solvent,the residue obtained was purified by column chromatography(dichloromethane/petroleum ether 1:4), yielding 5.25 g (66.8% yield) ofp-(o-carboranyl) benzyl(1,3-dithiane) [MS (EI) m/e 324.1 (M⁺); ¹H-NMR(CDCl₃) δ ppm: 1.6-3.3 (br, 10H, BH), 3.36 and 3.47 (m, 2H each,CH₂CH₂), 3.91 (br s, 1H, o-carborane-CH), 5.56 (s, 1H, SCH), 7.40 (d,2H, ArH, J=8.1 Hz), 7.46 (d, 2H, ArH, J=8.1 Hz)]. To a solution of thelatter compound (5.00 g, 15.43 mmol) in 5% aqueous THF (25 mL) underArgon, was added dropwise a solution of HgClO₄ (12.50 g, 31.29 mmol) inTHF (15 mL). The final mixture was stirred at room temperature for 15minutes, before being filtered and the precipitate washed 3 times with25 mL of diethyl ether. The filtrate was washed with an aqueoussaturated solution of Na₂CO₃ (3×25 mL) and with water (2×25 mL), beforebeing dried over anhydrous Na₂SO₄. The residue obtained afterevaporation of the solvent was purified by column chromatography(dichloromethane/petroleum ether 1:4) to give the title compound (3.27g, 85.6% yield); MS (EI) m/e 248.2 (M⁺); ¹H-NMR (CDCl₃) δ ppm: 1.60-3.2(br, 10H, BH), 4.03 (br s, 1H, o-carborane-CH), 7.65 (d, 2H, ArH, J=8.4Hz), 7.86 (d, 2H, ArH, J=8.4 Hz), 10.04 (s, 1H, CHO).

[0060] Synthesis of Compound 13:

[0061] meso-tetra[4-(o-carboranyl)phenyl]porphyrin (13): A solution ofaldehyde 12 (1.05 g, 4.23 mmol) and freshly distilled pyrrole (0.30 mL,4.32 mmol) in dry dichloromethane (430 mL) was purged with Argon for 30minutes. TFA (0.20 mL, 2.52 mmol) was added to the solution and thefinal mixture was stirred at room temperature under Argon for 24 hours.After addition of p-chloranil (0.780 g, 3.14 mmol) the final reactionmixture was stirred at room temperature for 3 hours. The solution wasconcentrated under vacuum to 300 mL, then washed once with an aqueoussaturated solution of NaHCO₃, and once with water before being driedover anhydrous Na₂SO₄. After evaporation of the solvent under vacuum,the resulting residue was purified by column chromatography(dichloromethane/petroleum ether 1:2) and the fastest running porphyrinfraction was collected and recrystallized from dichloromethane/methanol,yielding 0.220 g (17.7% yield) of the title compound as purple crystals,m.p.>300° C.; MS (MALDI) m/e 1184.5 (M+1); ¹H-NMR (CDCl₃) δ ppm: −2.89(br, 2H, NH), 1.7-3.5 (br, 40H, BH), 4.28 (br s, 4H, o-carborane-CH),7.89 (d, 8H, ArH, J=8.0 Hz), 8.17 (d, 8H, ArH, J=8.0 Hz), 8.78 (s, 8H,β-H). ¹H-NMR (d-TFA/CDCl₃) δ ppm: −0.97 (br, 4H, NH), 1.8-3.4 (br, 40H,BH), 4.31 (br s, 4H, o-carborane-CH), 8.13 (d, 8H, J=8.0 Hz), 8.51 (d,8H, J=8.0 Hz), 8.68 (s, 8H, β-H). UV-Vis (CHCl₃) λ_(max): 418 nm (ε464,700), 514 (17,165), 550 (8,300), 590 (5,635), 646 (4,035).

[0062] Synthesis of Compound 14:

[0063] meso-tetra[4-(nido-carboranyl)phenyl]porphyrin tetrapotassiumsalt (14): Porphyrin 13 (0.0500 g, 0.0423 mmol) was dissolved in a 3:1mixture of pyridine and piperidine (4.0 mL), and stirred at roomtemperature in the dark for 36 hours, under Argon. The solvent wascompletely removed under vacuum, the residue re-dissolved in a 60%acetone aqueous solution and passed slowly through a Dowex 50W2-100resin in the potassium form. The porphyrin fraction was collected, driedunder vacuum, re-dissolved in a 30% acetone aqueous solution and againpassed through the ion-exchange resin. After removal of the solventunder vacuum, the tetraanionic porphyrin was recrystallized frommethanol/diethyl ether, yielding 0.0494 g (90.2% yield) of the titlecompound, m.p.>300° C. ¹H-NMR (CD₃COCD₃) δ ppm: −2.78 (s, 2H, NH), −2.45to −1.90 (br, 4H, BH), 0.8-2.4 (br, 32H, BH), 2.57 (br s, 4H,nido-carborane-CH), 7.66 (d, 8H, ArH, J=8.0 Hz), 7.97 (d, 8H, ArH, J=8.0Hz), 8.87 (s, 8H, β-H). UV-Vis (acetone) λ_(max): 420 nm (ε 302,900),516 (11,560), 554 (10,580), 594 (3,335), 650 (4,875).

[0064] Synthesis of Compound 15:

[0065] Zn(II)-meso-tetra[4-(o-carboranyl)phenyl]porphyrin (15): To asolution of porphyrin 13 (0.085 g, 0.072 mmol) in dichloromethane (50mL), THF (4.0 mL), and pyridine (0.5 mL) was added ZnCl₂.2H₂O (0.30 g),and the final mixture was stirred at room temperature under Argonovernight. The mixture was then washed once with water, dried overanhydrous Na₂SO₄, and the solvent evaporated under vacuum. The residuewas purified by column chromatography (dichloromethane/cyclohexane 1:2),the pink color fraction collected and recrystallized fromdichloromethane/methanol, to give 0.085 g (94.7% yield) of the titlecompound as purple crystals, m.p.>300° C.; MS (MALDI) m/e 1246.7 (M⁺);¹H-NMR (CDCl₃) δ ppm: 1.6-3.6 (br, 40H, BH), 4.30 (br s, 4H,o-carborane-CH), 7.91 (br s, 8H, ArH), 8.17 (br s, 8H, ArH), 8.88 (br s,8H, β-H). UV-Vis (CH₂Cl₂) λ_(max): 424 nm (ε 607,400), 554 (22,566), 594(6,781).

[0066] Synthesis of Compound 16:

[0067] Zn(II)-meso-tetra[4-(nido-carboranyl)phenyl]porphyrintetrapotassium salt (16): The Zn(II) complex 15 (0.0600 g, 0.0481 mmol)was dissolved in a 3:1 mixture of pyridine and piperidine (4.0 mL), andstirred at room temperature in the dark for 36 hours, under Argon. Thesolvent was completely removed under vacuum, the residue re-dissolved ina 60% acetone aqueous solution and passed slowly through a Dowex50W2-100 resin in the potassium form. The porphyrin fraction wascollected, dried under vacuum, re-dissolved in a 30% acetone aqueoussolution and again passed through the ion-exchange resin. After removalof the solvent under vacuum, the tetraanionic porphyrin wasrecrystallized from methanoldiethyl ether, yielding 0.0615 g (94.0%yield) of the title compound; ¹H-NMR (CD₃COCD₃) δ ppm: −2.54 to −1.78(br, 4H, BH), 0.6-2.2 (br, 32H, BH), 2.58 (br s, 4H, nido-carborane-CH),7.64 (d, 8H, ArH, J=8.1 Hz), 7.95 (d, 8H, ArH, J=8.1 Hz), 8.88 (s, 8H,β-H). UV-Vis (acetone) λ_(max): 426 nm (ε 432,000), 558 (14,380), 598(9,513).

[0068] Synthesis of Compound 17:

[0069] 3-Ethynylbenzaldehyde (17): To a solution of 3-bromobenzaldehyde(10.00 g, 54.08 mmol) and triphenylphosphine (0.500 g, 1.91 mmol) inanhydrous triethylamine (100 mL) under Argon, were addedethynyltrimethylsilane (6.00 g, 61.09 mmol) and palladium (II) acetate(0.100 g, 0.445 mmol). The final mixture was heated to reflux for 2hours, and then it was cooled down to room temperature and filtered. Thefiltrate was concentrated under vacuum to a thick oil, which waspurified by column chromatography (dichloromethane/petroleum ether 1:4)to give 8.52 g (78.0% yield) of 3-(trimethylsilylethynyl)benzaldehyde[¹H-NMR (CDCl₃) δ ppm: 0.26 (s, 9H, SiMe₃), 7.47 (t, 1H, ArH, J=7.5 Hz),7.70 (d, 1H, ArH, J=7.5 Hz), 7.82 (d, 1H, ArH, J=7.5 Hz), 7.96 (s, 1HArH), 9.98 (s, 1H, CHO)]. This compound (5.00 g, 24.74 mmol) was treatedwith K₂CO₃ (0.500 g) in methanol (50 mL) at 25° C. for 2 hours, underArgon. The solvent was removed under vacuum and the residue dissolved indichloromethane (100 mL). This solution was washed once with an aqueoussaturated solution of NaHCO₃ and once with water, before being driedover anhydrous Na₂SO₄ and the solvent evaporated under vacuum. Theyellow residue was purified by column chromatography usingdichloromethane/petroleum ether 1:4 for elution and recrystallizationfrom cyclohexane to give 2.80 g (87.1% yied) of the title compound; MS(EI) m/e 130.0; (M⁺). ¹H-NMR (CDCl₃) δ ppm: 3.20 (s, 1H, CH), 7.51 (t,1H, ArH, J=7.8 Hz), 7.74 (d, 1H, ArH, J=7.8 Hz), 7.87 (d, 1H, ArH, J=7.8Hz), 7.99 (s, 1H, ArH), 10.02 (s, 1H, CHO).

[0070] Synthesis of Compound 18:

[0071] 3-(o-carboranyl)benzaldehyde (18): BF₃.OEt₂ (0.11 g, 0.77 mmol)was added at 0° C. and under Argon, to a solution of3-ethynylbenzaldehyde (17) (1.00 g, 7.69 mmol) and 1,2-ethanedithiol(0.73 g, 7.75 mmol). This mixture was stirred at room temperature underArgon for 15 minutes. The reaction mixture was then washed once with a10% aqueous NaOH solution, once with an aqueous saturated solution ofNaHCO₃ and once with water, before being dried over anhydrous Na₂SO₄ andthe solvent evaporated under vacuum. Purification of the resultingresidue by column chromatography (dichloromethane/petroleum ether 1:4)gave 1.28 g (80.7% yield) of m-ethynylbenzyl(1,3-dithiane) [MS (EI) m/e206.0 (M⁺); ¹H-NMR (CDCl₃) □ ppm: 3.07 (s, 1H, CH), 3.37 and 3.50 (m, 2Heach, CH₂CH₂), 5.59 (s, 1H, SCH), 7.27 (t, 1H, ArH J=7.8 Hz), 7.38 (d,1H, ArH, J=7.8 Hz), 7.50 (d, 1H, ArH, J=7.8 Hz), 7.66 (s, 1H, ArH)].Decaborane (0.0500 g, 4.10 mmol), ethyl sulfide (0.750 g, 8.32 mmol) anddry toluene (25 mL) were combined in a schlenk tube equipped with a stirbar. This solution was heated at 40° C. for 3 hours and at 60° C. for 2hours, and then allowed to cool down to room temperature. To thismixture was added a solution of m-ethynylbenzyl(1,3-dithiane) (0.800 g,3.88 mmol) in dry toluene (5 mL), and the final reaction mixture wasslowly warmed up to 80° C. and stirred at this temperature for 3 days.After cooling to room temperature, the mixture was concentrated undervacuum and the resulting oil was dissolved in methanol (100 mL) andheated to reflux until liberation of hydrogen subsisted (approximately60 minutes). At room temperature a 50% aqueous HCI solution (1.0 mL) wascautiously added and the mixture was again heated to reflux until thehydrogen evolution was complete (approximately 30 minutes). Aftercooling down to room temperature, the reaction mixture was diluted withethanol and the excess ethylsulfide was removed by ethanol-ethylsulfideco-distillation. The remaining residue was concentrated under vacuum. Toa solution of the resulting residue in benzene (50 mL) at 5° C., wasadded 100 mL of a cold 10% aqueous NaOH solution, and the final mixturestirred vigorously for 15 minutes. The organic layer was separated,washed with water (3×15 mL) and dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the residue obtained was purified by columnchromatography (dichloromethane/petroleum ether 1:4), yielding 0.905 g(72.0% yield) of m-(o-carboranyl)benzyl(1,3-dithiane) [¹H-NMR (CDCl₃) δppm: 1.40-3.20 (br, 10H, BH), 3.39 and 3.49 (m, 2H each, CH₂CH₂), 3.97(br s, 1H, o-carborane-CH), 5.58 (s, 1H, SCH), 7.28 (t, 1H, ArH, J=7.8Hz), 7.39 (d, 1H , ArH, J=7.8 Hz), 7.55 (d, 1H, ArH, J=7.8 Hz), 7.63 (s,1H, ArH)]. To a solution of the latter compound (1.00 g, 3.09 mmol) in5% aqueous THF (10 mL) under Argon, was added dropwise a solution ofHgClO₄ (2.50 g, 6.26 mmol) in THF (5.0 mL). The final mixture wasstirred at room temperature for 15 minutes, before being filtered andthe precipitate washed with 25 mL of diethyl ether. The filtrate wasthen washed with an aqueous saturated solution of Na₂CO₃ (3×10 mL) andwith water (2×10 mL), before being dried over anhydrous Na₂SO₄. Theresidue obtained after evaporation of the solvent was purified by columnchromatography (dichloromethane/petroleum ether 1:4) to give the titlecompound (0.677 g, 88.5% yield); ¹H-NMR (CDCl₃) δ ppm: 1.5-3.3 (br, 10H,BH), 4.04 (br s, 1H, o-carborane-CH), 7.56 (t, 1H, ArH, J=7.8 Hz), 7.79(d, 1H, ArH, J=7.8 Hz), 7.91 (d, 1H, ArH, J=7.8 Hz), 7.96 (s, 1H, ArH),10.02 (s, 1H, CHO).

[0072] Synthesis of Compound 19:

[0073] meso-tetra[3-(o-carboranyl)phenyl]porphyrin (19): A solution ofaldehyde 18 (0.702 g, 2.83 mmol) and freshly distilled pyrrole (0.200mL, 2.88 mmol) in dry dichloromethane (285 mL) was purged with Argon for30 minutes. TFA (0.100 mL, 1.26 mmol) was added to the solution and thefinal mixture was stirred at room temperature under Argon for 18 hours.After addition of p-chloranil (0.522 g, 2.10 mmol) the final reactionmixture was stirred at room temperature for 3 hours. The solution wasconcentrated under vacuum to 200 mL, then washed once with water, oncewith an aqueous saturated solution of NaHCO₃, and again once with waterbefore being dried over anhydrous Na₂SO₄. After evaporation of thesolvent under vacuum, the resulting residue was purified by columnchromatography (dichloromethane/petroleum ether 1:2) and the fastestrunning porphyrin fraction was collected and recrystallized fromdichloromethane/methanol, yielding 0.140 g (16.7% yield) of the titlecompound as purple crystals; MS (MALDI) m/e 1184. ¹H-NMR (CDCl₃) δ ppm:−2.88 (br, 2H, NH), 1.6-3.5 (br, 40H, BH), 4.19 (br s, 4H,o-carborane-CH), 7.78 (m, 4H, ArH), 7.94 (m, 4H, ArH), 8.27 (m, 4H,ArH), 8.33 (m, 4H, ArH), 8.80 (s, 8H, β-H),UV-Vis (CHCl₃ Imax: 418 nm,512, 549, 588, 644.

[0074] Synthesis of Compound 20:

[0075] meso-tetra[3-nido-carboranyl)phenyl]porphyrin tetrapotassium salt(20): Porphyrin 19 (0.010 g, 0.008 mmol) was dissolved in a 3:1 mixtureof pyridine and piperidine (4.0 mL), and stirred at room temperature inthe dark for 36 hours, under Argon. The solvent was completely removedunder vacuum, the residue re-dissolved in a 60% acetone aqueous solutionand passed slowly through a Dowex 50W2-100 resin in the potassium form.The porphyrin fraction was collected, dried under vacuum, re-dissolvedin a 30% acetone aqueous solution and again passed through theion-exchange resin. After removal of the solvent under vacuum, thetetraanionic porphyrin was recrystallized from methanol/diethyl ether,yielding 0.0108 g (98.1% yield) of the title compound, m.p.>300° C.1H-NMR (CD₃COCD₃) d ppm: −2.70 (s, 2H, NH), −2.40 to −1.90 (br, 4H, BH),0.8-2.3 (br, 32H, BH), 2.48 (br s, 4H, nido-carborane-CH), 7.49 (m, 4H,ArH), 7.65 (m, 4H, ArH), 7.84 (m, 4H, ArH), 8.15 (m, 4H, ArH), 8.88 (s,8H, b-H). UV-Vis (acetone) Imax: 416 nm (e 326,300), 512 (14,300), 547(8,000), 590 (4,300), 646 (4,300).

[0076] Synthesis of Compound 21:

[0077] Bis-(3,5-bromomethyl)bromobenzene (21): To a refluxing solutionof 3,5-dimethylbromobenzene (4.63 g, 25.0 mmol) in dry CCl₄ (300 mL)under Argon, were added NBS (9.79 g, 55.0 mmol) and benzoyl peroxide(0.80 g, 3.30 mmol) in portions, over a one hour period. The finalreaction mixture was refluxed with stirring and under Argon for 16hours. After cooling to room temperature, the reaction mixture wasfiltered and the filtrate washed once with an aqueous saturated solutionof NaHCO₃ and once with water. The organic solution was dried overanhydrous Na₂SO₄ and the solvent evaporated under vacuum. The resultingresidue was purified by column chromatography usingdichloromethane/petroleum ether 1:9 for elution, and the main productcollected and recrystallized from n-hexane, yielding 2.83 g (33% yield)of the title and literature known compound; ¹H-NMR (CDCl₃) δ ppm: 4.40(s, 2H, CH2), 7.34 (s, 1H), 7.47 (s, 2H).

[0078] Synthesis of Compound 22:

[0079] Bis[3,5-(1-methyl-o-arboranyl)methyl]bromobenzene (22): n-BuLi(5.2 mL, 1.6 M in hexane) was added dropwise to a solution ofmethyl-o-carborane (1.39 g, 8.80 mmol) in dry THF (80 mL), at atemperature between −5° and 0° C., under Argon. The mixture was stirredat this temperature range for one and a half hours, then cooled to−15°-−20° C. (ice/salt bath). A solution of LiI (0.166 g, 1.27 mmol) indry THF (15 mL) and a solution of compound 21 (1.37 g, 4.00 mmol) in dryTHF (20 mL) were added and the final reaction mixture allowed to warm upto room temperature and stirred for 16 hours. After quenching thereaction with water the resulting mixture was extracted with diethylether. The organic extracts were washed once with water, once withbrine, dried over anhydrous Na₂SO₄ and the solvent removed under vacuum.The crude product was purified by column chromatography usingdichloromethane/petroleum ether 2:8 for elution, and the main productcollected and recrystallized from n-hexane to give 1.26 g (63% yield) ofthe title compound; MS m/e 497.3; ¹H-NMR (CDCl₃) δ ppm: 1.4-3.0 (br,20H, BH), 2.17 (s, 6H, CH₃), 3.43 (s, 4H, CH₂), 6.96 (s, 1H), 7.31 (s,2H).

[0080] Synth sis of Compound 23:

[0081] Bis[3,5-(1-methyl-o-carboranyl)m thyl]benzaldehyde (23): Asolution of compound 22 (0.994 g, 2.00 mmol) in THF (20 mL) under Argonwas cooled to −78° C. n-BuLi (1.4 mL, 1.6 M in hexane) was addeddropwise via syringe. After stirring the reaction mixture for 30 minutesat −78° C., dry DMF (0.77 mL, 10.x mmol) was slowly added. The resultingyellow mixture was stirred at −78° C. for 30 minutes and then warmed to0° C. and stirred at this temperature for one hour. temperature. A 5%aqueous HCl solution was added until the pH of the reaction mixture wasbetween 2 and 3, and the final mixture stirred at room temperature for30 minutes. The aqueous layer was extracted 4 times with diethyl ether,the organic fraction dried over anhydrous MgSO₄ and the solventevaporated under vacuum. Purification by column chromatography(dichloromethane/petroleum ether 2:3), afforded the title compound(0.632 g) in 70.9% yield; MS m/e 446.4; ¹H-NMR (CDCl₃) δ ppm: 1.5-3.0(br, 20H, BH), 2.20 (s, 6H, CH₃), 3.55 (s, 4H, CH₂), 7.30 (d, 1H, J=1.6Hz), 7.67 (d, 2H, J=1.6 Hz), 10.03 (s, 1H, CHO).

[0082] Synthesis of Compound 24:

[0083] meso-tetra[bis-3,5-(1-methyl-o-carboranyl)methylphenyl]porphyrin(24): A solution of aldehyde 23 (0.243 g, 0.54 mmol) and freshlydistilled pyrrole (0.050 mL g, 0.72 mmol) in dry dichloromethane (55 mL)was purged with Argon for 15 minutes. TFA (0.03 mL, 0.377 mmol) wasadded and the final solution was stirred at room temperature overnight(until complete disappearance of the starting aldehyde and formation of2 new spots by TLC). After oxidation with p-chloranil (0.102 g, 0.41mmol) for 6 hours at room temperature, the final reaction mixture waswashed once with an aqueous saturated solution of NaHCO₃ and once withwater, before being dried over anhydrous Na₂SO₄. The residue obtainedafter removal of the solvent was purified by column chromatography usingdichloromethane/petroleum ether 1:2 for elution. The porphyrin fractionobtained was recrystallized from dichloromethane/methanol, to give 0.30g (12% yield) of the title compound; MS m/e 1977.3 ¹H-NMR (d-TFA/CDCl₃)δ ppm: −0.80 (br, NH), 1.5-3.1 (br, 80H, BH), 2.31 (s, 24H, CH₃), 3.91(s, 16H, CH₂), 7.72 (s, 4H), 8.33 (s, 8H), 8.74 (s, 8H, β-H).

[0084] Synth sis of Compound 26:

[0085] 5,15-bis[bis-3,5-(1-methyl-o-carboranyl)methylphenyl]porphyrin(26): A solution of aldehyde 23 (0.446 g, 1.00 mmol) and dipyrromethane25 (0.146 g, 1.00 mmol) in dry dichloromethane (100 mL) was purged withArgon for 15 minutes and cooled down to 0° C. TFA (0.05 mL, 0.629 mmol)was added to the solution and the final mixture was stirred at 0° C. for2 hours and then at room temperature overnight. After oxidation withp-chloranil (0.277 g, 1.13 mmol) for 6 hours at room temperature, thefinal reaction mixture was washed once with an aqueous saturatedsolution of NaHCO₃, once with water, and once with brine, before beingdried over anhydrous MgSO₄. The residue obtained after removal of thesolvent was purified by column chromatography (alumina) usingdichloromethane for elution. The porphyrin fraction obtained wasrecrystallized from acetone to give 33.6% yield (0.192 g) of the titlecompound; MS m/e 1144.0; ¹H-NMR (d-TFA/CDCl₃) δ ppm: −1.92 (br, NH),1.4-3.2 (br, 40H, BH), 2.30 (s, 12H, CH₃), 3.89 (s, 8H, CH₂), 7.71 (s,2H), 8.34 (s, 4H) β-H, J=4.5 Hz), 9.61 (d, 4H, β-H, J=4.5 Hz), 10.98 (s,2H, meso-H).

[0086] Synthesis of Compound 27:

[0087] 5,15-bis[bis-3,5-(1-methyl-nido-carboranyl)methylphenyl]porphyrintetrapotassium salt (27): Porphyrin 26 (0.100 g, 0.087 mmol) wasdissolved in a 3:1 mixture of pyridine and piperidine (4.0 mL), andstirred at room temperature in the dark for 36 hours, under Argon. Thesolvent was completely removed under vacuum, the residue re-dissolved ina 60% acetone aqueous solution and passed. slowly through a Dowex50W2-100 resin in the potassium form. The porphyrin,fraction wascollected, dried under vacuum, re-dissolved in a 30% acetone aqueoussolution and again passed through the ion-exchange resin. After removalof the solvent under vacuum, the tetraanionic porphyrin wasrecrystallized from methanol/diethyl ether, yielding 0.102 g (92.8%yield) of the title compound. ¹H-NMR (CD₃COCD₃) δ ppm: −2.84 (s, 2H,NH), −2.45 to −1.85 (br, 4H, BH), 0.9-2.4 (br, 32H, BH), 1.66 (s, 12H,CH₃), 3.52 (s, 8H, CH₂), 7.67 (s, 1H, ArH), 7.74 (s, 1H, ArH), 8.37 (s,4H, ArH), 9.53 (dd, 4H, β-H), 9.61 (dd, 4H, β-H), 10.58 (s, 2H, meso-H).UV-Vis (acetone) λ_(max): 406 nm (ε 312,600), 502 (13,400), 536 (7,800),576 (6,100), 630 (3,100).

[0088] Synthesis of Compound 28:

[0089]Zn(II)-5,15-bis[bis-3,5-(1-methyl-o-carboranyl)methylphenyl]porphyrin(28): To a solution of porphyrin 26 (0.065 g, 0.057 mmol) indichloromethane (100 mL) and THF (10 mL), was added ZnCl₂.2H₂O (0.031 g,0.288 mmol), and the final mixture was stirred at room temperature underArgon overnight. The mixture was then washed once with water, dried overanhydrous Na₂SO₄, and the solvent evaporated under vacuum. The residuewas purified by column chromatography (dichloromethane/cyclohexane 2:1),the pink color fraction collected and recrystallized fromdichloromethane/methanol, to give 0.061 g (89.x % yield) of the titlecompound; MS (MALDI) m/e 1206.6. ¹H-NMR (CDCl₃) δ ppm: 1.4-3.0 (br, 40H,BH), 2.11 (s, 12H, CH₃), 3.64 (s, 8H, CH₂) 7.33 (s, 2H, ArH), 7.98 (s,4H, ArH), 8.98 (d, 4H, β-H, J=4.5 Hz), 9.41 (d, 4H, β-H, J=4.5 Hz),10.25 (s, 2H, meso-H).

[0090] Synthesis of Compound 29:

[0091]Zn(II)-5,15-bis[bis-3,5-(1-methyl-nido-carboranyl)methylphenyl]porphyrintetrapotassium salt (29): The Zn(II) complex 28 (0.050 g, 0.041 mmol)was dissolved in a 3:1 mixture of pyridine and piperidine (4.0 mL), andstirred at room temperature in the dark for 36 hours, under Argon. Thesolvent was completely removed under vacuum, the residue redissolved ina 60% acetone aqueous solution and passed slowly through a Dowex50W2-100 resin in the potassium form. The porphyrin fraction wascollected, dried under vacuum, re-dissolved in a 30% acetone aqueoussolution and again passed through the ion-exchange resin. After removalof the solvent under vacuum, the tetraanionic porphyrin wasrecrystallized from methanol/diethyl ether, yielding 0.052 g (94.8%yield) of the title compound; ¹H-NMR (CD₃COCD₃) δ ppm: −2.45 to −1.85(br, 4H, BH), 0.9-2.4 (br, 32H, BH), 1.65 (s, 12H, CH₃), 3.50 (s, 8H,CH₂), 7.66 (s, 1H, ArH), 7.73 (s, 1H, ArH), 8.28 (s, 4H, ArH), 9.41 (dd,4H, β-H), 9.47 (dd, 4H, β-H), 10.33 (s, 2H, meso-H). UV-Vis (acetone)λ_(max): 412 nm (ε 263,100), 496 (1,400), 542 (9,900), 580 (1,070), MS(MALDI) m/e 1319.4.

[0092] Synthesis of Compound 30:

[0093] The Zn(II) complex of carboranyl compound 30 of FIG. 7, bearingfour hydrophobic o-carboranes and four hydrophilic 3-hydroxypropyl sidechains, was prepared from dipyrromethane 31 and benzaldehyde 23, in 28%overall yield. The benzyl ester groups of 31 were removed by catalytichydrogenation (H₂—Pd/C), followed by decarboxylation usingtrifluoroacetic acid (TFA). Condensation of the resulting1,9-diunsubstituted dipyrromethane with benzaldehyde 23, usingLindsey-type conditions (TFA as the acid catalyst and p-chloranil as theoxidizing agent), produced a tetra-propionic ester porphyrin in 40%yield. Metalation of this compound using zinc (II) chloride indichloromethane, at room temperature, quantitatively produced thecorresponding Zn(II) porphyrin. Reduction of the methyl ester groupswith 5.2 equivalents of lithium aluminum hydride in tetrahydrofuran at0° C., gave the Zn(II) complex of porphyrin 30 in 70% yield. The Zn(II)complex of 30 is soluble in methanol but only partially soluble inwater.

[0094] In Vitro and In Vivo Analysis of Boronated Porphyrins

[0095] In vitro studies using rat 9L gliosarcoma cells, mouse B16melanoma cells and human U-373MG glioblastoma cells have been performed.We found that all compounds studied have very low dark cytoxicities, arereadily taken up and retained by cells and localize in specific cellorganelles, mostly in close proximity to the cell nucleus.

[0096] Our biological results to date indicate that these compounds havevery low in vivo toxicities. So far we have determined maximum tolerateddoses (MTD) for 8 of our compounds using healthy female Balb/c mice, andfor all compounds tested we found MTD>300 mg per Kg of body weight.

[0097] We have also done a biodistribution study with 2 of ourcompounds, using male Fisher 344 rats bearing 9L glioma tumors, andachieved tumor to normal brain boron concentration ratios of 5 andhigher. This compares favorably with drugs currently in BNCT clinicaltrials—BSH and BPA—which both show lower selectivities, i.e. smallerratios.

[0098] Cytotoxicity/Phototoxicity Assays

[0099] Human glioblastoma U373 MG and mouse melanoma B16 cells wereobtained from ATCC. The rat gliosarcoma line was kindly provided by theUCSF Brain Tumor Research Group. All cells were maintained in log phasemonolayer cultures with RPMI 1640 supplemented with 10% fetal bovine and2 mM glutamine.

[0100] Cells were seeded in 96-well culture plates, allowed to settleand attach for 24-48 hours, and then triplicate wells were exposed totwo fold serial dilutions of test compounds at concentrations up to 250μM. Compounds 4, 6, 10, and the Zn(II) complex of 10 in crystalline formwere carefully weighed and dissolved in 100% DMSO to prepare stocksolutions; subsequent dilutions were done directly into the culturemedium just prior to administration to cells. After short term (2 hours)or long term (24-48 hours) exposure, cells were washed and wellsrefilled with fresh culture medium. For dark toxicity trials cells wereallowed to proliferate for an additional 48-72 hours. For phototoxicitytrials, washed cells were irradiated for 10 minutes with broad spectrum(600-700 nm) red light and then returned to an incubator for 48-72hours.

[0101] Exposure to drug for 2 hours followed by drug wash-out did notinhibit proliferation of any of the three cells types. Exposure for 24hours was inhibitory only at higher concentrations (IC₅₀-≧150 μM) for 9Land U-373 MG cells, but B16 viability was unaffected. Whereas the metalfree porphyrins 4 and 10 display nearly identical IC₅₀ values (˜150 μM)in the affected cells, their Zn(II) complexes were found to be about 20%less toxic (IC₅₀˜180-185 μM), Thus, the nido-carboranylporphyrincompounds of the present invention display low dark toxicity.

[0102] For carboranylprorphyrin 4, irradiation with broad spectrum lightcaused cytotoxicity at 2 hours (IC₅₀=50 μM) and 24 hours exposure(IC₅₀=1.5 μM). As observed for the dark toxicity experiments, thecorresponding Zn(II) complex 6 was about 20-fold less phototoxic. Thesephototoxicity results indicate that the carboranylporyphin compounds ofthe present invention would be active in PDT. As with dark toxicity, B16cells were more resistant that the other two cell types.

[0103] Cellular Uptake and Retention

[0104] The concentration-dependent uptake of compounds 4, 6, 10 and thecorresponding Zn(II) complex of 10 was investigated in cells exposed to1, 5, and 10 μM of the porphyrins for 24 hours. The concentration ofintracellular-bound porphyrin was determined by chemical extraction ofwashed cell monolayers, followed by spectrophotometric and/or IMP-MSdeterminations. The uptake values for 9L and U-373MG cells were verysimilar and exceeded that of B16 cells. The porphyrin accumulationinvariably increased with increasing exogenous drug concentration andthe uptake of nido-carboranylprophyrins 4 and 10 was approximately4-times greater than that of the corresponding Zn(II) complexes.

[0105] The uptake of carboranylporphyrins by log phase cells was alsoshown to be time-dependent. Cells exposed to 5 μM drug concentrationscontained increasing amounts of extractable porphyrin over the 24-houruptake period examined in these studies. While 9L and U-373MG cells hadsimilar uptake levels, B16 cell cultures consistently accumulated 60-70%less drug on a per cell basis. Cell-bound porphyrin that could not beremoved by rinsing the cells with Hanks balanced salt solution (HBSS)was detectable as early as 1 hour after introducing drug to the cellculture. In experiments using 9L cell s exposed to a 10 μM concentrationof free-base porphyrins 4 and 10, intracellular levels >60 μg of boronper billion cells (or gram of wet tissue) were achieved, following a 24hour exposure to drug.

[0106] Intacellular Localization

[0107] Confocal fluorescence microscopy was used to examine theintracellular localization of nido-carboranylporphyrin 4 in live cells.Rat 9L tumor cells and human normal keratinocyte line HaCaT were used inthese studies. HaCaT cells were included in these particular experimentsbecause they adhere and spread nicely on glass cover slips, thusfacilitating the imaging process. Cells exposed to 2 μM concentration ofporphyrin 4 for either 6 or 24 hours were examined at a magnification of200× for intracellular fluorescence using excitation/emissionwavelengths optimized for this type of compound. The 9L and HaCaT cellsshowed a similar intracellular fluorescent pattem. In both cases, 100%of the cells were labeled and the cells exposed to drug for 24 hourswere slightly brighter than those exposed for 6 hours. The punctuatefluorescence was predominantly perinuclear, with many cells having anadditional local area of concentration that appeared to be adjacent tothe nuclear membrane. No fluorescent signal was detectable in the plasmamembranes of isolated individual cells nor in the intercellularjunctions of confluent cells.

[0108] In Vivo Biodistribution

[0109] Male Fisher 344 rats with bilateral subcutaneous 9L tumors wereinjected i.v. (2.2 ml) with a 2 mM solution of carboranylporphyrin in 4%Cremophorlsaline. Groups of three rats each were sacrificed at 2, 8, 18,24 and 48 hours following drug administration. None of the rats showedany signs of discomfort or toxicity from injection of thenido-carboranylporphyrins. Plasma, tumor, and normal brain tissue boronlevels were determined by IMP-MS. Rapid elimination of boron from plasmawas observed between 2 and 8 hours. Boron concentrations in normal brainwere always lower than those in tumor and a tumor to brain concentrationratio of 6 was seen at 8 hours following drug administration.

BIBLIOGRAPHY

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What is claimed is:
 1. A porphyrin compound wherein a carboranyl groupis attached to the porphyrin compound by a carbon-carbon linkage.
 2. Theporphyrin compound of claim 1 corresponding to general formula I:

wherein M is 2H or a divalent metal ion; R1 and R2 are eachindependently hydrogen, alkyl or hydoxyalkyl; and R3 through R6 arehydrogen or a phenyl group of general formula II:

wherein R7 through R11 are hydrogen, a hydrophilic group or a carboranylgroup, said carboranyl group being attached to the phenyl group by acarbon-carbon linkage; and either one or two of R7 through R11 arehydrophilic or said carboranyl groups; and at least one of R3 through R6is a phenyl group of general formula II having at least one of saidcarboranyl groups.
 3. The compound of claim 2 wherein said carboranylgroup is 1-methyl-o-carboranyl or o-carboranyl.
 4. The compound of claim1 wherein the compound is at least about 15% boron by weight.
 5. Thecompound of claim 4 wherein the compound is about 25% to about 44% boronby weight.
 6. The compound of claim 1 wherein two of R3 through R6 arephenyl groups of general formula II, each having at least one of saidcarboranyl groups.
 7. The compound of claim 1 wherein R3 through R6 arephenyl groups of general formula II, each having at least one of saidcarboranyl groups.
 8. The compound of claim 1 wherein the hydrophilicgroup is hydroxyl, NMe₃ ⁺, PMePh₂ ⁺, PO(OH)₂, SO₃H, COOH or NH₂.
 9. Amethod of making a porphyrin compound comprising the steps of: a)providing a reaction mixture comprising a pyrrole or a dipyrrole, and abenzaldehyde dissolved in a suitable solvent, wherein the pyrrole ordipyrrole is unsubstituted or substituted with alkyl groups and thebenzaldehyde is of general formula III

 wherein R7 through R11 are each hydrogen, a hydrophilic group or acarboranyl group, said carboranyl group being attached to thebenzaldehyde by a carbon-carbon linkage; and b) subjecting the reactionmixture to an acidic pH until the benzaldehyde and the pyrrole or thebenzaldehyde and the dipyrrole are converted to a porphyrin compound.10. The method of claim 9 wherein the reaction mixture is subjected toan acidic pH by the addition of TFA.
 11. The method of claim 9 furthercomprising oxidation of the reaction mixture with tetrachloroquinone ordichlorodicyanobenzoquinone.
 12. The method of claim 9 furthercomprising complexing the porphyrin compound with a divalent metal ion.13. The method of claim 9 further comprising forming a salt of theporphyrin compound.
 14. The method of claim 9 wherein the solvent isdichloromethane.
 15. The method of claim 9 wherein the carboranyl groupis 1-methyl-o-carboranyl or o-carboranyl.
 16. The method of claim 9wherein the hydrophilic group is hydroxyl, NMe₃ ⁺, PMePh₂ ⁺, PO(OH)₂,P(CH₂OH)₂, SO₃ ⁻, COOH, CO₂ ⁻ or NH₂.
 17. An intermediate for use in thesynthesis of porphyrin compounds comprising a benzaldehyde of generalformula III

wherein R7 through R11 are each hydrogen, a hydrophilic group or acarboranyl group, said carboranyl group being attached to thebenzaldehyde by a carbon-crbon linkage; and either one or two of R7through R11 are said carboranyl groups.
 18. The intermediate of claim 17wherein the carboranyl group is 1-methyl-o-carboranyl or o-carboranyl.19. The intermediate of claim 17 wherein the hydrophilic group ishydroxyl, NMe₃ ⁺, PmePh₂ ⁺, P(CH₂H)₂, SO₃ ⁻, COOH or CO₂ ⁻.
 20. A methodof delivering an effective amount of a neutron capture agent to tumorcells comprising contacting the tumor cells with the porphyrin compoundof claim
 1. 21. The method of claim 20 wherein the porphyrin compound isadministered to a patient and selectively taken up by the tumor cells.22. The method of claim 20 wherein the tumor cells are brain tumor ormelanoma cells.
 23. The method of claim 21 wherein the amount ofporphyrin compound taken up by the tumor cells is an effective amount ofthe compound, wherein an effective amount is an amount sufficient forcytotoxicity when said tumor cells are irradiated by thermal neutrons.24. A method of delivering an effective amount of a photosensitizingagent to tumor cells comprising contacting the tumor cells with theporphyrin compound of claim
 1. 25. The method of claim 24 wherein theporphyrin compound is administered to a patient and selectively taken upby the tumor cells.
 26. The method of claim 24 wherein the tumor cellsare brain tumor or melanoma cells.
 27. The method of claim 25 whereinthe amount of porphyrin compound taken up by the tumor cells is aneffective amount of the compound, wherein an effective amount is anamount sufficient for cytotoxicity when the tumor cells are irradiatedby red light.
 28. A composition for use in boron neutron capture therapycomprising an effective amount of the porphyrin compound of claim 1, anda pharmaceutically acceptable carrier or excipient, wherein theeffective amount is an amount sufficient for selective uptake, retentionand damage to tumor cells when said tumor cells are irradiated bythermal neutrons.
 29. A composition for use in photodynamic therapycomprising the porphyrin compound of claim 1 and a pharmaceuticallyacceptable carrier or excipient wherein the effective amount is anamount sufficient for selective uptake, retention and cytotoxicity totumor cells when said tumor cells are irradiated by red light.